NO is a free radical and is now thought to be an important intracellular signaling molecule. It in low concentrations, can act as a pro-oxidant presumably by forming peroxynitrite with superoxide, whereas at higher concentrations its antioxidant action predominates. There is convincing evidence that cellular pro-oxidant states-i.e., increased concentrations of active oxygen species and organic peroxides and radicals can promote initiated cells to neoplastic growth. On the other hand, pro- oxidant states can be prevented or suppressed by the enzymes of the cellular antioxidant defense system and many antioxidants are anti-promoters and anti-carcinogens. It is, therefore, not surprising that NO has been shown to involve cellular transformation and stimulation of neoplastic growth, whereas other studies indicate that NO has a cytostatic and/or cytotoxic effect on tumor cells. The overall hypothesis to be tested is that "NO has a biphasic action on human breast cancer cell lines, i.e. at low steady state concentrations, it increases cell proliferation, whereas at high steady state concentrations, it decreases cell proliferation. This biphasic action is due to its actions on cyclin D1-cyclin dependent kinase (CDK)-retinoblastoma protein (pRG) cascade. NO also acts as an intermediary in some of the actions of estradiol leading to cell proliferation." This hypothesis will be tested under 4 specific aims. They are as follows: Specific Aim 1. We will determine the effects of different concentrations of NO on cell cycle and proliferation and DNA synthesis of human breast cancer (HBC) cell lines. Specific Aim 2. We will determine the effects of low steady state concentrations of NO to modulate phosphorylation of pRB in HBC cell lines, increase cyclin D1 expression, and elucidate the step(s) at which NO may be acting on the cyclin D1-CDK-pRB cascade. Specific Aim 3. We will determine the effects of high steady state concentrations of NO (determined under specific aim 1) to modulate the phosphorylation of pRB and elucidate the step(s) at which NO may be acting on the cyclin D1-CDK-pRB cascade in HBC cell lines. Specific Aim 4. We will determine the ability of inhibitors of NO synthesis to modulate the estradiol induced increase in breast cancer cell proliferation in vitro; shorten the G0/G1 phase of the cell cycle and the molecular mechanisms involved in this phenomenon.